Regulatory proteins use the energy of ligand binding or other effectors to manipulate individual bonding interactions and larger scale structure changes with the purpose of producing changes in function. The true currency of these interactions is structural free energy. For example the archetypal allosteric protein, hemoglobin, controls the oxygen affinity of its heme groups by using part of the binding energy of its initial oxygen ligands to break particular bonding interactions that selectively stabilize its low affinity, T state. To understand how these energy interconversion processes work, it will be necessary to locate the binding interactions and structure changes that play an important role, measure their individual free energies, quantify in free energy terms how the different changes interact, and then see how it all fits together to construct the allosteric machinery. We have developed hydrogen exchange methods that can locate and quantify site-resolved free energy changes. These methods are being used to locate allosterically active bonding interactions in hemoglobin and, together with mutational and partial liganding approaches, to measure how the different bonds interact with each other and with the allosteric process in real free energy terms. This work has so far been applied to a limited but important part of the protein. Work is also being done to upgrade the hydrogen exchange analysis by adding a mass spectrometry component in order to extend the hydrogen exchange labeling technology to the whole protein. We propose to continue these studies and the new developmental efforts. This work will profit greatly by being placed in the context of a collaborative program project group devoted to parallel studies that use a wide range of structural, spectroscopic kinetic, and genetic methodologies.